PROJECT SUMMARY (30 lines) Alveolar rhabdomyosarcoma (ARMS) is a common and aggressive muscle cancer that affects hundreds of children annually in the United States. ARMS are pathognomonic with oncogenic chromosomal translocations between the PAX genes and the fork-head transcription factor (FOXO1). Yet, the pathways that the PAX3/7- FOXO1 transcription factors modulate, the tumor cells of origin, and the therapeutic vulnerabilities that arise in ARMS cells is still unclear, largely due to lack of precision animal models that accurately recapitulate the same translocation fusion events found in human ARMS. The long-term goal of our work is to uncover therapeutically relevant pathways that drive ARMS growth. The overall objective of this application is to develop zebrafish models of ARMS to identify therapeutic vulnerabilities that can be exploited clinically. Our central hypothesis is that worse survival outcomes in PAX3-FOXO1+ ARMS are reflected in differences in cells of origin, elevated numbers of molecular defined tumor-propagating cells (TPCs), and predisposition to metastasis. The feasibility of our approach is supported by our work in using zebrafish to model a wide range of human cancers, recent optimization of Crispr/CAS9 approaches to create patient-specific translocations using CRE/Lox, and our successful high-content imaging screening approach to identify FDA approved drugs with efficacy in curbing growth of other RMS subtypes. The rationale for our research is that there are few good experimental animal models that accurately mimic the underlying genetics of human ARMS, obviating our ability to define how specific oncogenic fusions drive cancer growth. This work is significant because it will uncover divergent cellular mechanisms that account for differences in the clinical manifestation of PAX3/7-FOXO1+ ARMS, identify new therapies to treat ARMS, and provide new modeling approaches for translocation+ cancers, all expressed goals outlined in PA-16-251 supported by the Cancer Moonshot Initiative. Aim 1 will characterize differences in PAX3/7-FOXO1-induced ARMS using innovative zebrafish models, testing our hypothesis that fusion+ ARMS have inherent differences in proliferation and cell(s)-of-origin. Aim 2 will assess PAX3/7-FRKH for differential effects on modulating metastasis and tumor propagating potential, testing our hypothesis that PAX3-FOXO1+ ARMS do worse clinically because they have elevated TPC numbers and metastatic capacity. Aim 3 will use an innovative high-content imaging screen to identify FDA-approved drugs that kill TPCs and suppress growth of human ARMS. With respect to outcomes, our research will develop much-needed precision animals models of ARMS and identify key differences in PAX3/7-FOXO1+ ARMS including likely differences in cell-of-origin, TPCs, and metastatic capacity, providing explanation of why PAX3-FOXO1+ ARMS do worse clinically. Our work will also identify novel therapies to kill TPCs in human patient derived xenografts (PDX). This work is expected to have a positive translational impact by demonstrating the pre-clinical efficacy of targeting TPCs in human ARMS and identifying new therapies for the treatment of this devastating cancer.